Comparative Transcriptome Analysis of CMV or 2b-Deficient CMV-Infected dcl2dcl4 Reveals the Effects of Viral Infection on Symptom Induction in Arabidopsis thaliana

Due to the impaired antiviral RNAi, the dcl2dcl4 (dcl2/4) mutant is highly susceptible to viruses deficient of the viral suppressor of the RNA silencing (VSR) contrast to wild-type Arabidopsis. It was found that more severe disease symptoms were induced in dcl2/4 infected with VSR-deficient CMV (CMV-Δ2b or CMV-2aTΔ2b) compared to wild-type Arabidopsis infected with intact CMV. In order to investigate the underlying mechanism, comparative transcriptome analysis was performed with Col-0 and dcl2/4 that were infected by CMV, CMV-Δ2b and CMV-2aTΔ2b, respectively. Our analysis showed that the systematic infection of CMV, CMV-Δ2b and CMV-2aTΔ2b could cause hypoxia response and reduce photosynthesis. Asymptomatic infections of CMV-Δ2b or CMV-2aTΔ2b in Columbia (Col-0) promoted the expression of cell division-related genes and suppressed the transcription of metabolism and acquired resistance genes. On the other hand, immunity and resistance genes were highly induced, but photosynthesis and polysaccharide metabolism-related genes were suppressed in diseased plants. More interestingly, cell wall reorganization was specifically caused in modestly diseased Col-0 infected by CMV and a strong activation of SA signaling were correspondingly induced in severely diseased dcl2/4 by CMV or CMV mutants. Thus, our research revealed the nature of the Arabidopsis–CMV interaction at the transcriptome level and could provide new clues in symptom development and antiviral defense in plants.


Introduction
Viruses are biotrophic parasites that must usurp host factors for their propagation. Pathogenesis usually occurs when plant virus overcomes plant defense to infect and causes detrimental effects to the host. Pathogenic viruses have a severely negative impact on agricultural production worldwide [1]. Establishing effective strategies for minimizing the damage of virus epidemics will require understanding the responses of plant hosts to viral infection. Given the broad host range and the detrimental effect on agriculture economy in worldwide, CMV is regarded as an important crop pathogen and also as a model virus to study plant-virus interactions [2]. CMV is commonly found in wild populations of Arabidopsis at up to 80% prevalence [3], and, therefore, the Arabidopsis-CMV interaction is relevant in nature.
Arabidopsis thaliana has been widely employed to investigate host responses to viruses because of its advantages for molecular genetic approaches. Through plenty of genetics analysis and function characterization on the infection of Arabidopsis by viruses, including but not limited to cucumber mosaic virus (CMV), turnip mosaic virus (TuMV), turnip yellow mosaic virus (TYMV), turnip crinkle virus (TCV) and cauliflower mosaic virus (CaMV), large amount of host factors with antiviral activity against plant viruses have been identified [4][5][6][7][8][9][10][11][12][13].
The RNAi-based antiviral defense is a fundamental antiviral innate immunity in plants to restrict virus replication and movement by slicing viral RNAs and inhibiting viral protein translation [14][15][16]. In the process, Dicer-like enzymes (DCLs) recognize long double-stranded viral RNAs as pathogen-associated molecules and process them into 21-24 nucleotide (nt) small interference RNAs (namely virus-derived siRNAs, vsiR-NAs) [17][18][19]. Subsequently, antiviral Argonaute proteins (AGOs) bind vsiRNAs to mediate either translational repression of complementary target mRNAs or transcriptional silencing of target DNAs [20]. In addition, two of six Arabidopsis RNA-dependent RNA polymerases (RdRPs), RDR1 and RDR6, efficiently synthesize double-stranded RNAs to generate secondary vsiRNAs through DCLs [21]. Thus, vsiRNAs are directly produced through the processing of DCLs. Arabidopsis has four DCLs, which function redundantly or cooperatively in the antiviral immunity [17,18]. DCL4 is the major player responding to the RNA virus in Arabidopsis, and DCL2 can subrogate DCL4 when the infected host is with the compromised DCL4 function [18]. In this context, only plants with null mutation on both DCL2 and DCL4 are susceptible to infection with CMV or other plant viruses and develop severe disease symptoms [5,22].
To counteract host antiviral silencing, most viruses have evolved viral suppressors of RNA silencing (VSRs) independently. VSRs from different viruses target distinct steps of the antiviral defense, such as blocking vsiRNA biogenesis by binding to viral dsRNAs or interacting with host DCLs, DsRNA binding protein (DRB), RDRs or the suppressor of gene silencing 3 (SGS3), preventing AGO-vsiRNA/RISC formation or function by sequestrating duplex siRNAs or disturbing the functions of AGOs, and limiting the spread of the silencing signal by disrupting the movement of viruses and vsiRNAs [16,23]. As one of the earliest documented VSRs, CMV 2b could directly bind both double-stranded siRNA and AGOs to suppress RNA-directed DNA methylation and posttranscriptional gene silencing (PTGS) [24][25][26][27]. Besides, CMV 2b is a determinant of virulence and controls the severity of symptom in plants [28,29]. CMV 2b causes developmental anomalies in Arabidopsis, which phenocopies ago1-25 and ago1-27 [27]. CMV with 2b deletion results in the symptomless infection of wild-type tobacco and Arabidopsis but causes severe disease symptoms in mutants defected in vsiRNA biogenesis [10,30,31]. Thus, CMV-∆2b or CMV-2aT∆2b, two 2b deficient CMV, has been employed to screen critical components of the RNAi-dependent antiviral immunity [10,32,33]. These analogical symptoms of the antiviral RNAi defective mutants infected with CMV-∆2b or CMV-2aT∆2b imply that there should be commonly disturbed host factors that are affected by CMV independent of 2b and contributed to the disease symptoms. However, most studies focus on the elucidation of antiviral RNAi against viral infection pathways, and much less attention was paid to dissecting endogenous biological processes in host plants disturbed by CMV-∆2b or CMV-2aT∆2b infection.
In this work, we analyzed the transcriptome of Columbia (Col-0) and dcl2dcl4 infected with CMV, CMV-∆2b, or CMV-2aT∆2b. Gene Ontology (GO) enrichment with differential expression of genes (DEGs) in different virus-infected plant showed that CMV, CMV-∆2b and CMV-2aT∆2b could generally induce hypoxia stress response and inhibit photosynthesis or chloroplast organization in Arabidopsis. Our further analysis demonstrated that asymptomatic infections of CMV-∆2b or CMV-2aT∆2b in wild type accompanied by the elevated expression of mitosis-related genes and the diminished transcription of chemical homeostasis and acquired resistance genes. However, symptomatic infections induced bacterium immunity and inhibited photosynthesis and carbohydrate metabolism. Interestingly, we found that mildly symptomatic CMV-infected Col-0 specifically displayed the induction expression of cell wall reorganization genes but severely symptomatic infected dcl2/4 specifically displayed a strong activation of salicylic acid (SA) signaling. Thus, our findings shed light on the interaction among intact or VSR-deprived CMV and wild-type or antiviral RNAi-deficient plants for better understanding the underlying molecular mechanism.

Plant Growth and Virus Infection
Arabidopsis thaliana ecotype Columbia (Col-0) was used as wild type and mutant dcl2dcl4 (dcl2/4) was described previously [33]. After synchronized at 4 • C in dark for 2 days, Arabidopsis were grown on soil at about 24 • C with 16 h light, 8 h dark cycle. The light intensity is about of 5000 lx. The virus used in this study is the Fny strain of CMV and the mutant virus CMV-∆2b and CMV-2aT∆2b, which are deprived of 2b, was described previously [33]. Purified virions propagated in Nicotiana clevelandii were used as the inocula at the concentration of 10 µg/mL. Plants about 25 days after germination were inoculated with inocula by the mechanic rubbing method.

Mapping and Quantification Differentially Expressed Genes (DEGs)
All the clean reads from Col-0 group and dcl2/4 group were separately mapped back to the reference sequences (Arabidopsis_thaliana.TAIR10. 50 The FPKM was used to do PCA analysis by "FactoMineR" [34]. Genes that differentially expressed between the mock control plants and virus-inoculated plants were screened using DESeq2 [35]. The threshold of differentially expressed genes was set to: the upregulated gene as log 2 FoldChang > 1 and Padjust-value < 0.05; the down-regulated gene as log 2 FoldChange < −1 and Padjust-value < 0.05.

Gene Ontology (GO) Enrichment Analysis and Venn Analysis
Gene Ontology (GO) annotations of the contigs were determined using ClusterProfile [36] according to the molecular function, biological process, and cellular component ontologies (http://www.geneontology.org/ (accessed on 22 April 2022). GO-enrichment analysis of DEGs was performed using the ClusterProfile R pock [36], and the Padjust-value were calculated using the Benjamini-Hochberg (BH) correction. Compared to mock plants, genes with Padjust-value < 0.05 were included in our analysis. The overlapping induced or suppressed genes were analyzed (https://xlinux.nist.gov/dads/HTML/venndiagram. html (accessed on 7 May 2022).

Gene Expression Analysis
The systemically infected leaves of 10 to 15 plants were pooled for RNA extraction two weeks after inoculation. The first-strand cDNA was synthesized from 1 ug total RNA with HiScript@ II Q RT SuperMix for qPCR (R233-01, Vazyme) according to the user manual. Real time quantitative PCR (qPCR) was performed on a CFX 96 real-time PCR detection system (BioRad, Hercules, CA, USA) according to the Taq Pro Universal SYBR qPCR Master Mix (Q712-02/03, Vazyme). The PCR reactions were subjected to an initial denaturation step of 95 • C for 30 min, followed by 39 cycles of 95 • C for 15 s and 60 • C for 30 s. The actin2 gene was used as an internal control for studying the expression level of target genes. Relative gene expression levels were analyzed with 2-∆∆CT method [37]. All reactions were performed in three technical and biological replicates. The primers for target genes were designed respectively by qPCR Primer Database (https://biodb.swu.edu.cn/qprimerdb/, accessed on 13 May 2022). The detailed information of primers showed in Table S1.

Comparison of the Differential Transcriptome between CMV-Infected and Mock Plants
To identify differentially expressed genes (DEGs) responding to virus infections, we firstly compared the virus-inoculated group to the mock group using DESeq2 [35]. Compared to Col-Mock plants, 1850, 1338, 2111 genes were induced and 1385, 753, 1108 were suppressed in Col-CMV, Col-∆2b, and Col-2aT∆2b, respectively ( Figure 1c). Relatively, CMV-∆2b triggered less DEGs than CMV and CMV-2aT∆2b in wild type. Among these DEGs, 728 induced genes and 361 suppressed genes were overlapped in Col-0 group ( Figure 1d). Gene Ontology (GO) analysis showed that host genes related to hypoxia stress response (GO:0001666, response to hypoxia; GO:0036293, response to decreased oxygen levels; GO:0070482, response to oxygen levels; GO:0071456, cellular response to hypoxia; GO:0036294, cellular response to decreased oxygen levels; GO:0071453, cellular response to oxygen levels), cell cycle process (
The 376 induced genes enriched to anti-bacterium immunity (GO:0042742, defense response to bacterium; GO:1900426, the positive regulation of defense response to bacterium; GO:1900424, the regulation of defense response to bacterium; GO:0032103, the positive regulation of response to external stimulus; GO:0002237, response to molecule of bacterial origin) and ADP binding (GO:0043531) (Figure 3b, Table S3). Among all the induced genes that enriched in anti-bacterium immunity related functions, there are genes that have been reported to be involved in antiviral immunity. AGO2 (AT1G31280), one major antiviral RNAi AGO against RNA virus in Arabidopsis [31,[41][42][43][44], is significantly induced in symptomatic plants and maintained at low expression level in healthy Col-0 (Figure 3d,e). Except for the antiviral RNA silencing, resistance (R) gene-mediated immunity plays an important role in antiviral defense [45][46][47]. Typical R genes, which encode nucleotide-binding, leucine-rich repeat (NB-LRR) proteins, such as ACTIVATED DISEASE RESISTANCE 1 (ADR1, AT1G33560), PHYTOALEXIN DEFICIENT 4 (PAD4, AT3G52430) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1, AT1G73805) were also induced dramatically in diseased plants and only slightly induced in asymptomatic Col-∆2b and Col-2aT∆2b (Figure 3d,e). Notably, transcript levels of these four genes increased more dramatically in susceptible dcl2/4 (Figure 3e). The expression pattern of these four genes is in agreement with the symptom severity of virus-infected plants, suggesting a correlation between the CMV-induced phenotype and the expression of immunity and resistance genes.

Distinct Pathways Affected in Col-0 Infected by Intact CMV
With the removal of DEGs shown in dcl2/4 group, Col-CMV-sp had other 255 induced DEGs and 219 suppressed DEGs (Figure 3a), which were speculated to be the direct or indirect target of CMV 2b. The GO analysis of DEGs specifically regulated in Col-CMV showed that induced genes are related to polysaccharide metabolism (GO:0005976, polysaccharide metabolic process; GO:0044262, cellular carbohydrate metabolic process; GO:0044264, cellular polysaccharide metabolic process; GO:0000271, polysaccharide biosynthetic process; GO:0016051, carbohydrate biosynthetic process; GO:0010383, cell wall polysaccharide metabolic process; GO:0033692, cellular polysaccharide biosynthetic process; GO:0034637, cellular carbohydrate biosynthetic process; GO:0010410, hemicellulose metabolic process; GO:0006073, cellular glucan metabolic process), cell wall biogenesis (GO:0042546, cell wall biogenesis; GO:0071669, plant-type cell wall organization or biogenesis; GO:0009834, plant-type secondary cell wall biogenesis; GO:0009832, plant-type cell wall biogenesis; GO:0044036, cell wall macromolecule metabolic process) and ribosome (GO:0005840, ribosome; GO:0022626, cytosolic ribosome; GO:0044391, ribosomal subunit) ( Figure 4a, Table S4). These GO terms are involved in basic biological processes that are indispensable in cell wall formation. Genes related to cell wall, such as FASCICLIN-LIKE ARABINOGALACTAN-PROTEIN 11 (FLA11, AT5G03170) and EXPANSIN A20 (EXPA20, AT4G38210), were specifically induced in CMV infected Col-0 and dcl2/4 (Figure 4c,d). The induced DEGs infers that 2b protein may impact the synthesis of polysaccharide and cell wall biogenesis in wild-type Col-0.

Discussion
Transcriptome analysis offers a general view of affected pathways and development in virus-infected plants. Systematical infection with CMV or CMV mutants brings about some common physiological changes in host plants. Previous studies revealed that the virus infection can modify photosynthesis and disturb chloroplast components and functions [49][50][51][52], and coat protein of CMV is responsible for the chlorosis in infected leaves [51]. Indeed, we found numerous genes implicated in photosynthesis and chloroplast organization reduced expression pattern in CMV-infected plants ( Figures S2-S7). In addition, genes involved in cold response are also suppressed in all the six CMV infected plants ( Figures S2-S7). Considering that virus-triggered RNA silencing is inhibited at low temperature and CMVinfected plants increases tolerance to frost stress [53,54], there should be a crosstalk between cold response and virus infection.
In this work, genes related to hypoxia response are also consistently induced in all infected plants ( Figures S2-S7), which is manifested by the waterlogging phenotype of the systematically infected leaves (Figure 1a). The hypoxia response in systematically infected leaves may be due to the disturbance on the photoreaction and respiratory electron transport by virus infection [55]. It has been found that hypoxia tolerance required AGO1 and AGO4 RNA-silencing pathways [56][57][58][59], while virus infection silenced AGO1 [60]. Thus, the host plant may elevate hypoxia response to restore hypoxia tolerance upon viral infection.
As for the asymptomatic infection of CMV-∆2b or CMV-2aT∆2b to Col-0, lots of cell division related genes increased expression and genes involved in chemical homeostasis and acquired resistance were inhibited (Figure 2). We speculate that host plants reduce resistance and viruses promote cell division in hosts during the asymptomatic virus infection to achieve the mutualistic interaction between CMV-∆2b or CMV-2aT∆2b and Col-0. However, in symptomatic infected plants, bacterium immunity is elevated and cellular metabolism was reduced (Figure 3). The accumulation of AGO2 mRNA may be induced by the reduction of miR403 (Figure 3d, e) [61], which depends on the suppression of the AGO1 function by virus infection [60]. Enhanced AGO2 activation accompanies induced resistance to CMV in Arabidopsis [42,61,62]. Besides, SA-dependent defense is an effective antiviral strategy and responsible for the virus-caused symptoms [63]. Critical SA-related genes, PAD4, ADR1, and SARD1, are induced in the four diseased plants (Figure 3d,e). EDS1/PAD4 pathway promotes salicylic acid (SA) biosynthesis and maintains important SA-related resistance programs [64]. Activated by both surface-resident and intracellular LRR receptors, EDS1-PAD4-ADR1 signaling pathway acts as the convergence point for defense signaling cascades in pathogen immunity [65]. ADR1 exhibits resistance to a number of microbial pathogens, including CMV [66]. SARD1 is a key regulator for Isochorismate Synthase 1 induction and salicylic acid (SA) synthesis [67]. These induced resistance pathways may contribute to host survival upon virus aggression, and synchronously lead to the development of symptoms.
In modestly sick Col-0 infected with CMV, the cell wall component synthesis and cell wall related genes, FLA11 and EXPA20, which are associated with modification of cell wall [68,69], are specifically upregulated (Figure 4a,c,d). Regarded as a specific response to viruses, cell wall reorganization affects the spread of the virus by involving apoplast and symplast activation [70]. FLA11 and EXPA20 may take part in the cell wall reorganization in CMV-infected Arabidopsis in nature. The increased cell wall biogenesis may attenuate the developmental anomalies in Col-CMV, by contrast to the severely diseased phenotype of dcl2/4-CMV, dcl2/4-∆2b and dcl2/4-2aT∆2b.
PR proteins play important roles in response to abiotic and biotic stresses. Usually, the expressions of PRs are induced in systemic acquired resistance (SAR) or local acquired resistance (LCR) and minimize the accumulation of pathogens in uninfected plants organs [71][72][73][74]. Interestingly, the expression of PR1, PR2, PR4 and PR5 were increased drastically in virus-infected dcl2/4 but induced in a relative low extent in Col-CMV, and even reduced in Col-∆2b and Col-2aT∆2b (Figure 5d). The highest levels of PRs in dcl2/4-∆2b indicated the extreme induction of SA signaling, which should require pathogenicity but is independent of VSR 2b, in the RNAi-deficient plant. In addition, consistent with our above findings, the increased SA-mediated resistance would be related to the severe disease symptom in virus-infected dcl2/4 and could contribute to a major immunity for dcl2/4 survival under virus attack. Evidence also showed that disrupting NCER2 would induce higher levels of SA [75] and loss of NCER2 function can reduce Arabidopsis resistant to pathogens [76]. Indeed, the level of NCER2 significantly reduced in dcl2/4 and dcl2/4 was more susceptive to CMV compared with Col-0.
In summary, by comparing transcriptome sequence, we found that the systematic infection of CMV, CMV-∆2b and CMV-2aT∆2b cause hypoxia response and reduce photosynthesis. Cell wall organization and biogenesis-related genes are specifically induced in asymptomatic infected Col-∆2b and Col-2aT∆2b, which may facilitate the symbiosis of CMV mutants and Col-0. The symptom of Col-CMV, dcl2/4-CMV, dcl2/4-∆2b and dcl2/4-2aT∆2b is in correlation with the increased AGO2 and pathogen immunity. In addition, cell wall reorganization caused by CMV may relieve symptom development. Nevertheless, the strong activation of SA signaling might lead to severe symptoms in dcl2/4. Our research further revealed the interaction between plant Arabidopsis and CMV and provided clues in symptom development and antiviral defenses in plants. Further studies on the response of plant mutants in these biological processes would help to clarify and elucidate the mechanism underlying. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14071582/s1, Figure S1: Principal component analysis (PCA) of biological triplicates derived from RNA-seq; Figure S2: Gene ontology (GO) terms of differentially expressed genes (DEGs) in Col-CMV; Figure S3. GO terms of DEGs in Col-∆2b; Figure S4. GO terms of DEGs in Col-2aT∆2b; Figure S5. GO terms of DEGs in dcl2/4-CMV; Figure S6. GO terms of DEGs in dcl2/4-∆2b; Figure S7. GO terms of DEGs dcl2/4-2aT∆2b; Table S1. Primers used for Qpcr; Table S2. Analysis of transcriptome sequencing data of Arabidopsis-infected CMV; Table  S3. The log 2 Foldchange of DEGs in all diseased plants; Table S4. The log 2 Foldchange of DEGs specifically detected in Col-CMV; Table S5. The log 2 Foldchange of DEGs specifically detected in all virus-infected dcl2/4. Author Contributions: Conceptualization, Q.X. and Z.G.; methodology, L.S., F.C., M.W. and L.J.; validation, L.S. and L.J.; formal analysis, L.S. and Q.X.; writing-original draft preparation, Q.X.; writing-review and editing, Z.G. and Q.X.; project administration Z.G. and Q.X.; funding acquisition, Z.G. and Q.X. All authors have read and agreed to the published version of the manuscript.